Protein Kinase C Protocols

Protein Kinase C Protocols

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Since the discovery that protein kinase C (PKC) transduces the ab- dance of signals that result in phospholipid hydrolysis, this enzyme has been at the forefront of research in signal transduction. Protein Kinase C Protocols covers fundamental methods for studying the structure, function, regulation, subcellular localization, and macromolecular interactions of PKC. Protein Kinase C Protocols is divided into 11 sections representing the major aspects of PKC regulation and function. Part I contains an introduction and a historical perspective on the discovery of PKC by Drs. Yasutomi Nishizuka and Ushio Kikkawa. Part II describes methods to purify PKC. Part III describes the standard methods for measuring PKC activity: its enzymatic activity and its stimulus-dependent translocation from the cytosol to the membrane. Part IV describes methods for measuring the membrane interaction of PKC in vivo and in vitro. Part V provides methodologies and techniques for measuring the ph- phorylation state of PKC, including a protocol for measuring the activity of PKC's upstream kinase, PDK-1. Novel methods for identifying substrates are described in Part VI. Part VII presents protocols for expressing and analyzing the membrane targeting domains of PKC. Part VIII provides a comprehensive c- pilation of methods used to identify binding partners for PKC. Part IX describes pharmacological probes used to study PKC. The book ends with a presentation of genetic approaches to study PKC (Part X) and a discussion of approaches used to study PKC in disease (Part XI).
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Product details

  • Hardback | 584 pages
  • 158 x 232 x 32mm | 920.81g
  • Totowa, NJ, United States
  • English
  • 2003 ed.
  • XVI, 584 p.
  • 1588290689
  • 9781588290687
  • 2,584,763

Back cover copy

Protein kinase C impacts on nearly every aspect of signal transduction, placing it at the forefront of research in how cells transduce information. In Protein Kinase C Protocols, leading investigators from academia and industry present a comprehensive collection of classical and emerging laboratory techniques for the study of this influential protein, all optimized for successful and reproducible results. Described in step-by-step detail, these methods can be easily used to explore the structure, function, regulation, subcellular localization, and macromolecular interactions of protein kinase C. Included in this collection are easy-to-follow techniques to purify protein kinase C, to measure its activity and its membrane interaction in vivo and in vitro, and to identify substrates. There are also methodologies and techniques for measuring the phosphorylation state of protein kinase C, for expressing and analyzing the membrane-targeting domains of protein kinase C, and for detecting binding proteins. Additional chapters address pharmacological probes, genetic approaches, and protein kinase C in disease. Each protocol is introduced in the context of protein kinase C function and regulation and contains many notes on how best to deal with the problems that may occur.
Comprehensive and authoritative, Protein Kinase C Protocols offers a timely compilation of biophysical, biochemical, cell biological, and molecular biological approaches that will ensure successful results in every laboratory working in this rapidly advancing field.
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Table of contents

Part I. Introduction

The Ins and Outs of Protein Kinase C
Alexandra C. Newton

Early Studies of Protein Kinase C: A Historical Perspective
Yasutomi Nishizuka and Ushio Kikkawa

Part II. Expression and Purification

Expression and Purification of Protein Kinase C from Insect Cells
Hideyuki Mukai and Yoshitaka Ono

Expression and Purification of Protein Kinase Cd from Bacteria
Michael Gschwendt and Luise Stempka

Part III. Measuring the Activity of Protein Kinase C

Complexities in Protein Kinase C Activity Assays: An Introduction
Julianne J. Sando

Enzyme Assays for Protein Kinase C Activity
Julianne J. Sando and Jacquelyn K. Beals

Subcellular Translocation of Protein Kinase C
Julianne J. Sando, Jacquelyn K. Beals, and Isa M. Hussaini

Part IV. Measuring the Interaction of Protein Kinase C with Membranes

Measuring the Interaction of Protein Kinase C with Membranes: An Introduction
Alexandra C. Newton

Fluorescence Imaging of Protein Kinase C Translocation in Living Cells
Naoaki Saito

Measuring the Binding of Protein Kinase C to Sucrose-Loaded Vesicles
Jennifer Giorgione and Alexandra C. Newton

Use of Stopped-Flow Fluorescence Spectroscopy to Measure Rapid Membrane Binding by Protein Kinase C
Eric A. Nalefski and Alexandra C. Newton

[3H]Phorbol 12,13-Dibutyrate Binding Assay for Protein Kinase C and Related Proteins
Nancy E. Lewin and Peter M. Blumberg

Part V. Measuring the Phosphorylation of Protein Kinase C

Protein Kinase C Phosphorylation: An Introduction
Peter J. Parker

Pulse-Chase Analysis of Protein Kinase C
Mikiko Takahashi and Yoshitaka Ono

PDK-1 and Protein Kinase C Phosphorylation
Alex Toker

In Vitro Autophosphorylation of Protein Kinase C Isozymes
Antonio M. Pepio and Wayne S. Sossin

Tyrosine Phosphorylation of Protein Kinase C
Toshiyoshi Yamamoto, Emiko Yamauchi, Hisaaki Taniguchi, Hidenori Matsuzaki, and Ushio Kikkawa

Methods to Study Dephosphorylation of Protein Kinase C In Vivo
Karen England and Martin Rumsby

Phosphopeptide-Specific Antibodies to Protein Kinase C
Wayne S. Sossin

Part VI. Identifying Protein Kinase C Substrates

Identifying Protein Kinase C Substrates: An Introduction
Alex Toker

A Chemical Genetic Approach for the Identification of Direct Substrates of Protein Kinases
Kavita Shah and Kevan M. Shokat

Studying the Optimal Peptide Substrate Motifs of Protein Kinase C Using Oriented Peptide Libraries
Michael B. Yaffe

Part VII. Structural Analysis of Protein Kinase C

Structural Analysis of Protein Kinase C: An Introduction
James H. Hurley

Bacterial Expression and Purification of C1 and C2 Domains of Protein Kinase C Isoforms
Wonhwa Cho, Michelle Digman, Bharath Ananthanarayanan, and Robert V. Stahelin

Crystallization of the Protein Kinase Cd C1B Domain
Gongyi Zhang and James H. Hurley

Part VIII. Methods for Detecting Binding Proteins

Methods for Detecting Binding Proteins: An Introduction
Gerda Endemann and Daria Mochly-Rosen

Detection of Protein Kinase-Binding Partners by the Yeast Two-Hybrid Analysis
Betty Y. Chang and Christine A. Cartwright

Glutathione S-Transferase Pull-Down Assay
Deborah Schechtman, Daria Mochly-Rosen, and Dorit Ron

Overlay Method for Detecting Protein-Protein Interactions
Deborah Schechtman, Christopher Murriel, Rachel Bright, and Daria Mochly-Rosen

An Overlay Assay for Detecting Protein Kinase C-Binding Proteins and Substrates
Susan Jaken

Functional Proteomic Analysis of the Protein Kinase C Signaling System
Jason M. Pass, Jun
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Review quote

"...a comprehensive collection of established and emerging techniques used for the study of protein kinase C, and is quite simply an essential guide for any lab interested in cell signallin...an impressive collection of expert contributors...this is an excellent lab manual which provides very detailed and easy to follow methods and instructions that would be useful for any lab that is interested in any form of cell signalling..." - Proteomics



"...a valuable book for everybody interested in PKC, and it is a must for everybody doing research with it." - ChemBioChem
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